Propionibacterium freudenreichii culture optimization for vitamin B12 production


Jiménez Álamo, Carlos  


Vitamin B12, also called cobalamin, is a water-soluble vitamin that has an important role in cellular metabolism, acting as a cofactor for important enzymes present in the human body. The involvement of cobalamin in important processes such as the synthesis of DNA, fatty acids and myelin makes that its deficiency can lead to major health problems. For this reason, added to the aging society and the increase of diets that exclude animal products, its market value, and production necessity are increasing significantly. Typically, there are two different ways of large-scale vitamin production, chemically or by fermentation using microorganisms. In the particular case of B12 production, chemical synthesis is difficult and costly as a result of its complexity, making industrial production inviable. As a result, large-scale production is exclusively done by fermentation using selected non-genetically modified or genetically modified microorganisms. In this study, it has been used Propionibacterium freudenreichii subsp. shermanii NBRC12391, due to P. freudenreichii GRAS (Generally Recognized As Safe) status, as a production strain. Typically, the fermentation process uses Corn Steep Liquor (CSL) as a source of nitrogen, since it is a rich medium obtained as a by-product of corn wet milling which provides all the components needed for Propionibacterium growth and cobalamin production. However, CSL presents important disadvantages such as its composition is not exactly known and it presents a high batch-to-batch variability, hindering the control of the culture and the scale-up of the process. On the other hand, with a chemical-defined media (CDM), the composition is perfectly known, allowing to control of the exact nutritional requirements, being possible to carry out optimization processes and metabolic flux analysis for a better understanding of the strain and improve productivities. The main objective of this project is, therefore, to develop a chemically defined medium to optimize growth and production, replacing CSL as a source of nitrogen. With this purpose, an initial CDM was proposed based on works in which a CDM had been successfully developed for lactic acid bacteria (LAB), since it is known that they share an ecological niche with P. freudenreichii. Starting with the composition of the proposed medium, successive simplifications were performed based on Leave-one-out (LOO) strategy by fermentations in Falcon tubes, which increases culture throughput considerably. After, LOO results were validated in Erlenmeyer flasks. In this work, a simplified CDM equivalent to rich CDM was achieved, but with lower growth rates compared with CSL. Interestingly, with the new CDM P. freudenreichii is not able to produce propionic acid as the main fermentative by-product. Our main hypothesis is that one or more than one component that is needed to follow the Wood-Werkman pathway is not present in the CDM. Nevertheless, more work should be done to try to find it/them out. Once found, a Design of Experiences (DoE) could be defined to optimize growth and cobalamin production as without the Wood-Werkman pathway activity there was not cobalamin production. Finally, a simulation using SuperPro designer was carried out in order to evaluate and compare the viability of this process at an industrial scale for both CSL and CDM-based cultures. Results show that industrial-scale cultures in CDM can be more profitable despite having much lower growth rates.



Carnicer Heras, Marc
Pellicer Moya, Teresa  


IQS SE - Master’s Degree in Bioengineering